CN111410690B

CN111410690B - anti-CK 19 protein monoclonal antibody, cell line, preparation method and application thereof

Info

Publication number: CN111410690B
Application number: CN202010273740.8A
Authority: CN
Inventors: 高惠然; 杨清海; 陈惠玲; 周洪辉; 王小亚
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: Fuzhou Maixin Biotechnology Development Co ltd
Priority date: 2020-04-09
Filing date: 2020-04-09
Publication date: 2021-08-06
Anticipated expiration: 2040-04-09
Also published as: CN111410690A

Abstract

The invention relates to the field of biological detection, and provides an anti-CK 19 protein monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain variable regions are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The inventor also provides a preparation method of the anti-CK 19 protein monoclonal antibody, which selects a human CK19 amino acid SEQ ID NO.1 sequence, couples the sequence with KLH, adds a cysteine at the carboxyl terminal thereof and uses the cysteine as immunogen. The inventor also provides a hybridoma cell line for secreting anti-CK 19 protein, wherein the cell line is a mouse hybridoma cell line AbM58058-41A2-PU with the preservation number: CGMCC NO. 17489. The anti-CK 19 protein monoclonal antibody has high specificity and sensitivity, can specifically recognize cells expressing CK19 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

anti-CK 19 protein monoclonal antibody, cell line, preparation method and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an anti-CK 19 protein monoclonal antibody, a cell line, a preparation method and application thereof.

Background

CK19 has a molecular weight of 40kDa, and is mainly used for labeling various simple epithelia including glandular epithelia and is mainly used for diagnosing adenocarcinoma. The liver cells do not express CK19, so the liver cells can be used for identifying liver cancer and metastatic adenocarcinoma. The CK19 is mainly applied to diagnosis of 1 thyroid follicular papillary carcinoma and follicular carcinoma, 2 cholangiocellular carcinoma and hepatocellular carcinoma, and 3 chordoma and parachulioma.

Disclosure of Invention

The inventor provides an anti-CK 19 protein monoclonal antibody, wherein the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

Further, the monoclonal antibody specifically recognizes the CK19 protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID NO. 1.

Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO. 17489. The cell line is a mouse hybridoma cell line AbM58058-41A2-PU, and is classified and named as: a mouse hybridoma cell line which has been deposited at 3.04.2019 in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and which is prepared by the institute of microbiology, China academy of sciences, No.3, West Lu 1 Hospital, North Kyoho, Beijing, and the township.

Further, the anti-CK 19 protein is a mouse IgG2a subtype monoclonal antibody.

The inventor also provides a preparation method of the anti-CK 19 protein monoclonal antibody, which is characterized in that an amino acid sequence shown as SEQ ID NO.1 in CK19 protein is selected, and after a cysteine is added to the carboxyl terminal of the amino acid sequence, the amino acid sequence is coupled with carrier protein KLH to be used as immunogen.

The inventor also provides a hybridoma cell line for secreting anti-CK 19 protein, wherein the cell line is a mouse hybridoma cell line AbM58058-41A2-PU, the cell line is preserved in China general microbiological culture Collection center with the preservation number: CGMCC NO. 17489.

The inventor also provides the application of the anti-CK 19 protein monoclonal antibody in CK19 protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Different from the prior art, according to the combination structure of the CK19 protein and DNA, antigenicity, hydrophilicity and hydrophobicity of amino acids and secondary structure, the technical scheme selects an amino acid sequence KAALEDTLAETEARFGAQLAHIQA (SEQ ID No.1) region which is different from other similar proteins, has proper length and special antigenicity as an antigenic peptide. A cysteine is added at the carboxyl terminal of the conjugate, and KLH is coupled to serve as an immune source CK 19-KLH. Immunizing a mouse, and obtaining a monoclonal cell line AbM58058-41A2-PU capable of efficiently secreting the anti-CK 19 protein monoclonal antibody and an anti-CK 19 protein monoclonal antibody secreted by the cell line through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing CK19 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 shows the result of immunoblot detection of antibody secreted by AbM58058-41A2-PU hybridoma.

FIG. 2 is a graph showing the results of immunohistochemical staining of papillary thyroid cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 3 is a graph of immunohistochemical staining results for colon cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 4 shows the immunohistochemical staining results of gastric cancer sample 1 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

FIG. 5 shows the immunohistochemical staining results of gastric cancer sample 2 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right).

Detailed Description

EXAMPLE 1 preparation of immunogen

First, immunogen preparation

First, antigen polypeptide selection

Sequence and secondary structure analysis is carried out according to the protein sequence with the accession number of P08727 in Uniprot, and the molecular weight of the CK19 protein with the total length of 400 amino acids is about 44 kDa. According to the parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of the protein predicted by an online server http:// www.cbs.dtu.dk/services/NetSurfP/and through the analysis of its antigenicity index (Jameson BA, Wolf H. the antigenicity index: a novel homology for predicting antigenic derivatives. composite applied biosci.1988,4(1):181-6.), the amino acid sequence KAALEDTLAETEARFGAQLAHIQA near the C-terminus was selected as an antigen for chemical synthesis and for coupling, and a cysteine was added at the carboxyl-terminus of the polypeptide to provide thiol coupling.

Coupling and purification of polypeptide

The equine activated keyhole limpet hemocyanin kit from Thermo Scientific (cat # 77653) was selected and operated according to the protocol provided for the kit. For the polypeptide to be conjugated, the free thiol group in the polypeptide is first detected with Ellman's reagent (Thermo Scientific, cat # 22582): adding 100 mu L of Ellman reagent stock solution into a 96-well plate, adding 10 mu L of polypeptide solution, measuring the ultraviolet absorption value of the solution by a Nano Drop spectrophotometer under the condition that the lambda is 412nm, and performing the next step if the OD value is more than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide synthesis step for quality control. To begin conjugation, 200. mu.L of deionized water was added to each mcKLH package to make up a 10mg/mL solution of KLH, 2mg of hapten was dissolved in 500. mu.L of Imject EDC coupling buffer, 500. mu.L of polypeptide solution was added to 200. mu.L of carrier protein solution, 1mL of deionized water was added to one package of EDC (10mg) and shaken slowly to dissolve completely, 50. mu.L of polypeptide solution was added to mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment to give CK 19-KLH.

Example 2 establishment of AbM58058-41A2-PU hybridoma cell line

Immunization

CK19-KLH from example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), 4-6 week old female Balb/c mice (purchased from Beijing Wintolite laboratory animals technologies, Inc.) were immunized and 6 o' clock of each mouse were injected ventrally subcutaneously at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the multi-antibody titer of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the immunogen is uniformly mixed with normal saline, and the dosage is 50 mu g/mouse.

Secondly, cell fusion:

a mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Thirdly, cloning and ELISA screening positive hybridoma cells:

11 days after fusion, the size and density of the clone cell mass are moderate, round, solid and large clone masses (10 plates multiplied by 93) are sucked under a dissecting mirror and are injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture plate is placed into a culture medium with 37 ℃ and 5 percent CO₂Culturing in an incubator.

After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using CK 19-BSA. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen. Screening out hybridoma cell strain AbM58058-41A2-PU secreting specific monoclonal antibody. The cell strain is transferred into a T-75 culture flask to be amplified to a logarithmic growth phase for seed preservation or subsequent experiments are carried out.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, counted-5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

Identification of one, two subtypes

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2a, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 50 μ L of 0.15% ABTS (Southern Biotech) and 0.03% H/well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min. The results show that the monoclonal antibody of the invention is a murine monoclonal antibody of the IgG2a type.

Second, determination of affinity constant

CK19 polypeptide was coated at a concentration of 2. mu.g/ml, 100. mu.L/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ L per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a citric acid-phosphate buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 was added to each well to develop a color for 10min, and 50. mu.L of a 0.5M sulfuric acid solution was added to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of OD value corresponding to the dilution factor of the antibody, and finding out the dilution factor A corresponding to the 'platform OD value' of not less than 1/2. The affinity constant was calculated to be 7.68X10 using the following formula⁹。

III, monoclonal antibody reaction specificity and application effect

The immunoblotting method is used for detecting the recognition specificity of the monoclonal antibody, and the immunoblotting experiment process is as follows: each protein was loaded at about 5-10ng and subjected to 12% polyacrylamide gel electrophoresis. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibody CK19 (1: 1000 dilution) was added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane again with TBST, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDoc MP multicolor fluorescence imaging system (Bio-Rad), the result can be shown in the immunoblot detection result of AbM58058-41A2-PU hybridoma secretory antibody.

Example 5 tissue chip staining and characterization

First, chip preparation process

HE sections were first stained for each sample to identify tumor sites. And (5) drawing a circle on a tumor target site, and preparing to punch holes. When the blank receptor wax block is manufactured, a plastic frame is placed on a mold, molten paraffin (the melting point is 55-58 ℃) is poured into the mold, the mold is placed into a refrigerator with the temperature of-20 ℃ for 6min after being cooled to the room temperature, and the wax block is taken out of the mold. A sample needle with the diameter of 1mm is selected on a tissue sample machine to punch a hole on the receptor wax block, the hole depth is 3-4 mm, another punching needle with the diameter of 1mm is used for punching a hole on a marked part of the wax block to collect a tissue core, and the length of the tissue core is about 0.1mm shallower than the hole depth of the receptor wax block. The collected tissue core is inserted directly or carefully grasped with forceps into the hollow of the recipient wax block. The steps are repeated until the preparation of all sample points is completed. And finally, pressing all the tissue cores flat by using a glass slide to ensure that the wax block of the tissue core is flat and smooth. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 60 ℃ for 15min to enable the tissue core and the wax of the receptor wax block to be integrated, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30min at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6min, then taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into a refrigerator at 4 ℃ for storage for later use. Slicing continuously with thickness of 3 μm, rinsing in cold water, naturally spreading, transferring the slices into 45 deg.C warm water for 30 s, mounting with polylysine-treated glass slide, baking the obtained tissue chip in 65 deg.C oven for 2 hr, taking out, cooling at room temperature, and storing in-4 deg.C refrigerator.

Second, IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each time with 100%, 95%, 85% gradient ethanol hydration for 3 minutes each time and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. The PBS was spun off and the peroxidase blocker was added dropwise and incubated for 10 minutes at room temperature. Spin-drying the slices, adding diluted primary antibody (the dilution ratio of the antibody is designed according to the concentration of the antibody in the first dilution) at room temperature (25 deg.C), incubating for 1 hr, washing with PBS for 3X 3 min, adding secondary antibody at room temperature, incubating for 30min, washing with PBS for 3X 3 min, discarding PBS, and mixing with fresh solutionThe DAB color developing solution is placed for developing for 3-10 minutes. Hematoxylin counterstain for 20 seconds, PBS turns blue. Dehydration was carried out in a gradient of 85% (3 min), 95% (3 min), 100% (3 min) and 100% (3 min) in order, and finally two times xylene was cleared for 10min, followed by sealing with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the conditions of clear tissue staining distribution and accurate cell positioning, the staining results can be further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Thirdly, sample detection results:

the anti-CK 19 protein monoclonal antibody prepared by the technical scheme and the commercially available anti-CK 19 protein monoclonal antibody (murine monoclonal antibody A53-B/A2.26) are synchronously detected on 39 cases of papillary thyroid cancer, 42 cases of colon cancer, 36 cases of gastric cancer and 42 cases of liver cancer, and the results are shown in the following table.

FIG. 2 is a graph showing the results of immunohistochemical staining of papillary thyroid cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right). Among them, the CK19 staining results of AbM58058-41A2-PU were "+++" positive and commercially negative.

FIG. 3 is a graph of immunohistochemical staining results for colon cancer samples (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right). Among them, the CK19 staining results of AbM58058-41A2-PU were "+ + +" positive, and commercially negative.

FIG. 4 shows the immunohistochemical staining results of gastric cancer sample 1 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right). Among them, the CK19 staining results of AbM58058-41A2-PU were "+++" positive and commercially negative.

FIG. 5 shows the immunohistochemical staining results of gastric cancer sample 2 (CK 19 for AbM58058-41A2-PU on the left and commercial CK19 on the right). Among them, the staining results of CK19 of AbM58058-41A2-PU were "+++" positive, and commercially available ones were "+++" positive.

The result shows that the monoclonal antibody of the anti-CK 19 protein secreted by the AbM58058-41A2-PU cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the detection of positive tissues, the positive rate is obviously higher than that of the commercial CK19(A53-B/A2.26), which indicates that the sensitivity and the affinity of the anti-CK 19 protein monoclonal antibody prepared by the invention are higher than those of the commercial antibody. In the detection of liver cancer in negative tumor tissues, the detection result of the antibody is consistent with that of the commercially available antibody, which indicates that the specificity of the antibody is not different from that of the commercially available antibody.

The synchronous detection is carried out on 30 normal tissue chips, and the positive and negative results of the samples are consistent, which indicates that the specificity of the antibody in normal tissues is equivalent to that of the antibody on the market. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

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Claims

1. The anti-CK 19 protein monoclonal antibody is characterized in that the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes CK19 protein.

4. The monoclonal antibody according to claim 3, which specifically recognizes the amino acid sequence shown as SEQ ID No.1 in the CK19 protein.

5. A monoclonal antibody against CK19 protein is prepared from the hybridoma cell line whose preserving number is CGMCC No. 17489.

6. The monoclonal antibody of claim 1, wherein the anti-CK 19 protein is a mouse IgG2a subtype monoclonal antibody.

7. A hybridoma cell line for secreting monoclonal antibodies against CK19 protein is a mouse hybridoma cell line AbM58058-41A2-PU, and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO. 17489.

8. Use of the anti-CK 19 protein monoclonal antibody of any one of claims 1-6 in preparing a CK19 protein immunodetection reagent.

9. The use according to claim 8, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.